Early, accurate diagnosis of lysosomal storage disorders is a major challenge, even for trained specialists. Finding innovative, accurate diagnostic methods, and high throughput, cost-effective tools are crucial to medical progress and will contribute to improved quality of life. The goal of this work was to improve currently used protocols to determine activity of acid β-galactosidase, and discuss the possibility analysing lysosomal enzymes with microfluidic systems. A principle of the determination of β-galactosidase activity was fluorometric measurement of a deprotonated form of 4-methylumbelliferone released in the enzymatic reaction. Measurements were performed using Jurkat T cells as a source of the enzyme. We observed the temperature-dependent substrate inhibition effect and determined the substrate (4-MU-β-d-galactopyranoside) concentration which should be used to determine acid β-galactosidase activity at 37 °C (0.8 mM) and at room temperature (0.6 mM). We proved that the sample incubation time may be significantly reduced to only a few minutes. We also showed that the amount of alkaline buffer used to stop the enzymatic reaction may be minimized and even, in some cases, eliminated. The presented results show how the sensitivity of the available methods to diagnose patients suffer from gangliosidosis GM1 or Morquio B disease can be improved. The proposed method may be easily implemented with microfluidic systems, which currently are promising tools for point-of-care applications.